Role of the mammalian fibrinolytic system during Plasmodium sporozoite infection. After injection into the dermis by the mosquito bite, Plasmodium sporozoites must migrate through several barriers to invade the hepatocytes and continue its development. Recruitment of the mammalian host fibrinolytic protease plasmin is a mechanism used by several pathogens to enhance invasion and dissemination through physical barriers. Plasminogen is an abundant zymogen in mammals and is activated into the serine protease plasmin by tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Although the primary function of plasmin is the degradation of fibrin, it can also degrade extracellular matrix proteins that form physical barriers. Our data shows that Plasmodium sporozoites bind plasminogen, tPA and uPA from the host. Importantly, inhibition of plasminogen activation in mice uncovered multiple requirements of plasmin activity for sporozoite infectivity: 1) for sporozoite migration and escape from the skin, and 2) for sporozoite evasion of the complement system, and 3) for invasion of the liver. Our hypothesis is that Plasmodium sporozoites co-opt proteins from the host fibrinolytic system to degrade proteins from the extracellular matrix and from the complement system to facilitate motility in the skin and in the liver tissues, and for evasion of the innate immune system. Parasite receptors for plasminogen and tPA. Our data shows that Plasmodium sporozoites and gametes can bind plasminogen, tPA and uPA which results in plasminogen activation at the parasite surface. These three proteins presumably bind to parasite receptors. Once identified, these receptors can be targeted with antibodies or small molecules to block their interaction with the fibrinolytic proteins and therefore, inhibit parasite transmission. Surface enolase has been described as a receptor for human plasminogen; and surface GAPDH works as a receptor for plasminogen in various pathogens including bacteria, fungi, and parasites. Some plasminogen receptors can also be co-receptors for the plasminogen activators, e.g., enolase. In Plasmodium, the cell surface receptor for tPA is currently unknown. We hypothesized that parasite enolase and GAPDH, previously reported to occur on the surface of P. falciparum, are receptors for plasminogen and tPA. We observed that both, enolase and GAPDH, are detected on the surface of several developmental stages of the parasite, including micro- and macrogametes, zygotes, ookinetes, and sporozoites. Furthermore, we found that tPA and plasminogen bind non-competitively to both enolase and GAPDH in vitro. This binding is mediated by the kringle domains of both tPA and plasminogen. We are currently testing the potential of enolase and GAPDH as transmission-blocking targets, either individually or in combination. Role of the mosquito saliva in the activation of plasminogen. Increasing evidence show that the saliva of mosquitoes and other insect vectors, contains factors that enhance the virulence of the pathogens they transmit. Initial data shows that the saliva of Anopheles female mosquitoes contains a protein that activates tPA. Our hypothesis is that activation of tPA by the saliva will result in an increased activation of plasminogen at the biding site and consequently, an enhancement of sporozoite infectivity. The inactive t-PA exists in the form of a single chain molecule and the active t-PA is a cleaved product and results in two-chain t-PA. Using a fluorogenic substrate for tPA (D-Val-Leu-Lys-7-amido-4-methyl coumarin) we can detect activation of t-PA in the presence of salivary gland extract when compared to the control (no t-PA with salivary gland extract). Further, we show that boiling the salivary gland extract at 100C and 65C significantly decreased the t-PA activation suggesting that the salivary gland molecule that activates tPA is a protein. We were able to detect cleavage of single-chain tPA into two-chain tPA after incubation with salivary gland extracts which suggest that the tPA activator is a protease. Cleavage was not detected in the boiled salivary gland extract samples indicating loss of activity of the salivary gland extract. Our next goal is to identify the components of the salivary gland extracts which is responsible for activation of t-PA by fractionation of the salivary gland extract by size-exclusion chromatography. Transgenic mosquitoes expressing Human PAI to block malaria transmission. We have developed transgenic Anopheles mosquitoes that secrete human plasminogen activator inhibitor 1 (PAI-1) in their midgut and saliva. In humans, PAI-1 inhibits the activation of plasminogen by blocking the activity of tPA and uPA. Data from our lab shows that inhibition of plasminogen activation by PAI-1 inhibits parasite development in the mosquito midgut and transmission of sporozoites to the mammalian host. The rationale is that PAI-1 produced by these mosquitoes will inhibit plasminogen activation in the mosquito midgut lumen and at the biting site, thereby preventing parasite development and transmission to a new host. Each of the transgenic lines were analyzed for fitness, infectivity, and compared with its respective parental lines and a wild-type control (WT). No difference in survival was observed in sugar fed mosquitoes when comparing males and females mosquitoes from each line; except for the females of mosquitoes expressing huPAI in the midgut and in the midgut and saliva which survive 10 days longer than the parental and WT lines. Survival is even longer when the transgenic females are fed on a blood meal, surviving 20 days more than the control lines. When analyzed for fertility and fecundity, the transgenic lines do not differ from controls. These results are important as they show that the transgenic mosquitoes have a similar or better fitness than the wild type mosquitoes which could serve as an advantage for mosquito population replacement. We also tested the ability of the transgenic mosquitoes to inhibit malaria transmission using the mouse malaria Plasmodium berghei and the human malaria Plasmodium vivax. For P. berghei, the transgenic lines expressing huPAI in the midgut and/or in the saliva reduced the number of oocyst and sporozoites up to 95-100%, resulting in strong inhibition or blocking of transmission to mice. For P. vivax infections, the mosquitoes were fed on P. vivax infected new world monkeys (Saimiri boliviensis). Mosquitoes had a prevalence of infection of 7.51% in the line expressing huPAI in the midgut, 4.34% in the one expression this protein in the salivary glands and 1.96% in the line expressing huPAI in both tissues. When compared to the wild type mosquitoes, this effect represents an inhibition of 77.82% by the huPAI in the midgut, 88.36% in saliva, and 95.36% in the huPAI in both tissues. These data show that expression of huPAI-1 in mosquito midguts and saliva strongly inhibit parasite development in the mosquito and transmission to a new host and shows the potential of targeting the interaction between the malaria parasite and the fibrinolytic system as a promising target to develop new malaria interventions.